Ras oncogenes are frequently associated with cancer. Of the three isoforms of Ras, K-Ras is most often mutated in human tumors. Ras proteins are prototypical GTPases that are biologically active when associated with cellular membranes. K-Ras differs from the other isoforms in utilizing a polybasic region in conjunction with a farnesylated CAAX motif for association with the plasma membrane (PM). We have discovered that serine 181 within this polybasic region is a site for phosphorylation by PKC and that this modification neutralizes the positive charge to a sufficient degree to cause K-Ras to dissociate from the PM and translocate to internal membranes. The C-terminus of K-Ras thus constitutes a farnesyl-electrostatic switch regulated by PKC, analogous to the myristoyl-electrostatic switch of the MARCKS protein. Among the membranes for which phosphorylated, internalized K-Ras has affinity is the outer mitochondrial membrane. Moreover, we have found that K-Ras with a phosphomimetic residue at position 181 is a potent stimulator of apoptosis. These surprising new discoveries suggest a new strategy for interfering with the biological effects of oncogenic K-Ras by promoting its phosphorylation by PKC. As proof of principle we have shown that xenograft tumors in nude mice driven by oncogenic K-Ras are sensitive to the PKC agonist bryostatin 1 but tumors driven by oncogenic K-Ras that is phosphorylation-deficient are insensitive. Moreover, oncogenic K-Ras that lacks C-terminal phosphorylation sites is hyperactive in transforming cells suggesting a physiologic role for this modification in dampening K-Ras signaling. To extend these exciting preliminary findings we propose three aims: Specific Aim 1. Regulation of the farnesyl-electrostatic switch: we will develop quantitative assays for K-Ras translocation and study the regulation by PKC of the farnesyl-electrostatic switch. Specific Aim 2. Mechanisms of phospho-K-Ras mediated apoptosis: we will determine the molecular mechanism(s) through which phosphorylated K-Ras stimulates apoptosis. Specific Aim 3. The Role of C-terminal Phosphorylation of K-Ras in carcinogenesis: We test the hypothesis that the PKC agonist bryostatin 1 will have anti-neoplastic effects against K-Ras transformed human cells. We will also construct a knock-in mouse with a K-Ras allele that lacks C-terminal phosphorylation sites and test the hypothesis that such mice will have more aggressive tumors when models of carcinogen-induced, K-Ras dependent tumors are applied. These studies will not only elucidate a new and unexpected area of Ras biology but will also have immediate application for the quest to develop K-Ras specific therapies.